Liposomal composition containing mild acidic active agent

ABSTRACT

Provided is a method for preparing a liposomal composition. The method comprises the step of contacting a liposome solution with a mild acidic agent for a limited time. The liposome solution comprises a weak acid salt encapsulated within an aqueous interior space separated from the aqueous medium by a membrane comprised of a lipid mixture containing one or more lipids and a hydrophilic polymer conjugated lipid at a molar percentage of less than 3% based on the total amount of the lipid mixture. The time for encapsulating the agent to a desired amount at a predetermined ratio to lipids is dramatically reduced even under a condition without elevating the temperature to above ambient environment.

BACKGROUND 1. Technical Field

The present disclosure relates to a delivery vehicle, kit or method forpreparing liposomes encapsulating a therapeutic agent, particularly aweak acid drug.

2. Description of Related Art

There have been many approaches to improving the stability or otherproperties in association with therapeutic functions of a weak aciddrug. Weak acid drugs, such as prostaglandins (PGs), have disadvantagesfor pharmaceutical use. For example, Edex® is a sterile, pyrogen-freepowder containing in alpha-cyclodextrin inclusion complex, also known asan alpha-cyclodextrin form of prostaglandin E₁ (PGE₁-CD). It is freelysoluble in water and practically insoluble in ethanol, ethyl acetate andether. After reconstitution, the active ingredient, alprostadil,immediately dissociates from the α-cyclodextrin inclusion complex.However, due to the short half life in human body of PGE₁ (about 30seconds) and its serious side effect when being overdosed, a highfrequency of administrating limited dose remains necessary in treatmentof PGE₁-CD. Other pharmaceutical preparations are in the form ofsuspension in which PGE₁ is dissolved in lipid microspheres, which is anoil-in-water type emulsion. However, the stability on shelf or in plasmaof such preparations is not sufficient (U.S. Pat. No. 4,684,633).

In one aspect, to improve the inconvenient treatment of the conventionalweak acid drug preparations, liposomal formulations are applied todeliver weak acid drugs, such as PG. U.S. Patent Publication no.US20020182248 disclosed liposomal dispersion by using a specified lipid,sphingolipid. However, this conventional liposomal dispersion isprepared by passive loading with a non-ensured encapsulation efficiencyof PG. Unentrapped PG could lead to overdose problems during itsadministration to a subject.

In another aspect, to prevent aggregation of liposomes in blood and toavoid being captured by reticuloendothelial system (RES), the surface ofliposome is coated with poly(ethylene glycol)(PEG). PEG-derivatizedliposomes, known as stealth liposomes, that contain entrappeddoxorubicin show enhanced therapeutic efficacy in preclinical studiesdue to increased tumor tissue drug level achieved after treatment withlong-circulating liposomes (U.S. Pat. Nos. 5,013,556, 5,676,971).However, little is proven to be applicable for loading or encapsulatingweak acid drugs in PEG-derivatized liposomes with a prompt procedureunder ambient temperature for ease of clinical use. Moreover, there isstill a need of liposome dispersion having a high efficiency in loadingweak acid drugs, particularly PG, into PEG-derivatized liposomes toavoid problems of overdose caused by free drug or of degradation of drugin aqueous dispersion.

In yet another aspect, to stably encapsulate a chemical entity inliposomes at a higher efficiency, U.S. Pat. No. 5,939,096 utilizes aproton shuttle mechanism involving the salt of a chemical entity togenerate a higher inside/lower outside pH gradient and to achieve acation-promoted precipitation or low permeability across the liposometransmembrane barrier.

However, based on the previous technique for loading a weak acid druginto liposomes or stealth liposomes in conjunction with an experimentaldata (FIG. 1) conducted by the Applicants, it demonstrates that stealthliposomes possess poor encapsulation efficiency at an ambienttemperature (25° C.) and require a heating step to increase permeabilityof prostaglandins. The loading procedure requires elevating thetemperature above the transitional temperature of the liposomes,typically higher than 60° C., which is generally higher than an ambienttemperature. The elevated temperature accelerates the degradation oflabile drug, such as prostaglandins, when being exposed to an aqueousenvironment, and thus hampers the stability of the composition in longterm storage.

To overcome the shortcomings, the present disclosure provides a methodand a kit for preparing a liposomal composition containing polyethyleneglycol-derivatized liposomes adapted for obtaining a higherencapsulation efficiency at an ambient temperature and exerting adesired pharmacokinetics for sustained releasing a weak acid drug, andpreferably an enhanced stability in shelf storage to mitigate or obviatethe aforementioned problems.

SUMMARY

The present disclosure is based on the discovery that a reduced amountof hydrophilic polymer conjugated lipid in a mixture of lipids forforming liposomes with a gradient of a weak acid salt is useful forloading and retaining an acidic compound in liposomes. Accordingly, thepresent disclosure provides methods, kits, or compositions fordelivering a variety of acidic compounds useful in the diagnosis,prognosis, treatment or prevention of an illness, disease, condition orsymptom in a subject.

In one aspect of the present disclosure, provided is a delivery vehicle,said delivery vehicle comprising a liposome in an aqueous medium,wherein the liposome having an interior space, and

the interior space:

1) is aqueous,

2) is separated from the aqueous medium by a membrane comprised of alipid mixture, wherein the lipid mixture contains one or more lipids anda hydrophilic polymer conjugated lipid at a molar percentage of lessthan 3% based on the total amount of the lipid mixture; and

3) contains a weak acid salt.

In another aspect of the present disclosure, provided is a method ofpreparing a liposomal composition, which comprises:

contacting, in an aqueous medium, a liposome comprising a weak acid saltencapsulated within an aqueous interior space separated from the aqueousmedium by a membrane comprised of a lipid mixture;

with a mild acidic agent for a sufficient time, whereby the mild acidicagent becomes encapsulated within the liposome;

wherein the weak acid salt has a cation pairing with the mild acidicagent, and the lipid mixture contains one or more lipids and ahydrophilic polymer conjugated lipid at a molar percentage of less than3% based on the total amount of the lipid mixture.

In a group of embodiments, the hydrophilic polymer conjugated lipid ispolyethylene glycol-derived lipid at a molar percentage ranging from0.1% to 3% based on the total amount of the lipid mixture; preferablyfrom 0.5% to 3%; and more preferably, from 0.5% to 1%.

In a group of embodiments, the liposome further comprises a polyproticacid encapsulated within the liposome in the aqueous interior space,whereby the polyprotic acid provides for excellent retention of theentrapped prostaglandin in the liposome. Generally, the polyprotic acidis a natural acid or a synthetic biocompatible acid. Preferably, thepolyprotic acid is an organic tribasic acid. In one embodiment, thepolyprotic acid is selected from the group consisting of citric acid,succinic acid, tartaric acid or a combination thereof.

In a group of embodiments, the contacting in an aqueous medium, aliposome with a mild acidic agent is performed under a condition ofbeing at an ambient temperature for the time sufficient for the mildacidic agent becoming encapsulated within the liposome.

In yet another aspect of the present disclosure, provided is a kit forpreparing a liposomal composition containing a mild acidic agent, whichcomprises:

one container accommodating a lyophilized cake, wherein the lyophilizedcake contains a mild acidic agent; and

another container accommodating a liposomal solution, wherein theliposome solution contains a liposome in an aqueous medium,

wherein the liposome includes a weak acid salt encapsulated within anaqueous interior space separated from the aqueous medium by a membranecomprised of a lipid mixture, wherein the lipid mixture contains one ormore lipids and a hydrophilic polymer conjugated lipid at a molarpercentage less than 3% based on the total amount of the lipid mixture.

In one general embodiment, the mild acidic agent is a physiologicallyactive lipid derived from fatty acid. Particularly, the mild acidicagent is arachidonic acid metabolite, such as a prostaglandin (PG)including prostacyclin or a thromboxane, which is a hormone-likesubstance that participates in a wide range of body functions such asthe contraction and relaxation of smooth muscle, the dilation andconstriction of blood vessels, control of blood pressure, inhibition ofplatelet aggregation and modulation of inflammation. Prostaglandins havebeen developed as pharmaceuticals or therapeutic compound in thetreatment of hypertension, thrombosis, asthma, and gastric andintestinal ulcers, for indication of labor and abortion in pregnantmammals, and for prophylaxis of arteriosclerosis.

In one general embodiment, the mild acidic agent is prostaglandin A₁(PGA₁), prostaglandin A₂ (PGA₂), prostaglandin E₁ (PGE₁), prostaglandinE₂ (PGE₂), prostaglandin F₁α (PGF₁α) or prostaglandin F₁α (PGF₂α).

In one preferred embodiment, the mild acidic agent in accordance withthe present disclosure is Prostaglandin E₁ (PGE₁) (also known asalprostadil) or its derivative, such as but not limited to6-keto-Prostaglandin E₁, 15-keto-Prostaglandin E₁, 13,14-dihydro-15-ketoProstaglandin E₁, or 16,16-dimethyl-6-keto prostaglandin E₁, which issuitable for the treatment for intermittent claudication patients. Forintermittent claudication patients, alprostadil can increase theirperipheral blood flow and permeability of blood vessel, and inhibitplatelet aggregation. PGE₁ can relieve patients from pain caused byinsufficient blood flow in peripheral circulation. However, theconventional treatment with PGE₁-CD (alpha-cyclodextrin form) isinconvenience for patients in a regimen of BID (twice a day) for weeks.In another aspects, for half life of PGE₁ is short (varying betweenabout 30 seconds and 10 minutes) in human body and its side effect couldbe serious when overdosed (it reduces blood pressure), high frequencyfor limited dose is necessary in PGE₁-CD treatment.

For improving this inconvenience treatment of conventionalprostaglandins, the present disclosure provides a delivery vehicle, akit or a method for preparing a liposomal composition containingprostaglandin, particularly a liposomal PGE₁ formulation wherein PGE₁ isencapsulated within liposomes. One of the benefits for liposomalformulations is to allow PGE₁ gradually released from liposomes in theliposomal composition for treatments in subject diseases or symptoms atextended intervals. Also, since only free form PGE₁ will cause the sideeffect but not liposomal form, the dosage of liposomal PGE₁ can beincreased without gaining serious side effect problem. The deliveryvehicles, kits and methods according to the present disclosure provide amore friendly and continence product for patient usage.

One of the objectives of the present disclosure is to establish aready-to-use liposomal composition containing the mild acidic agent suchas PGE₁ in a form of two-vial kit. To achieve better clinical usage,higher encapsulation efficiency after a short duration and remoteloading at ambient temperature are required. Higher encapsulationefficiency reduces free form of the agent which could cause undesiredside effect while overdosed. On the other hand, more retained agentafter in vitro release, which represents sustained release properties,is also required. Besides, for such objective of the present disclosure,the kit should be stable in storage for at least one year in 4° C. andshow efficacy in animal models. Based on those requirements, theparameters of the subject delivery vehicle, kit and method are modifiedto achieve the requirements.

Other objectives, advantages and novel features of the disclosure willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrating a diagram of comparison between the encapsulationefficiency of prostaglandin into liposome with conventionally workableamounts of polyethylene glycol-derived lipid, 6%, under conditions ofambient temperature and elevated temperature; and

FIG. 2 illustrating a diagram of encapsulation efficiency versus atitration of polyethylene glycol-derived lipid in liposomal compositionobtained according to Example 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definition

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

As used herein, the singular forms “a”, “an” and “the” include theplural reference unless the context clearly indicates otherwise.

All numbers herein may be understood as modified by “about.”

Liposome

The term “liposome” as used herein is usually characterized by having anaqueous interior space sequestered from an outer medium by a membrane ofone or more bilayers forming a vesicle. Bilayer membranes of liposomesare typically formed by lipids, i.e. amphiphilic molecules of syntheticor natural origin that comprise spatially separated hydrophobic andhydrophilic domains. Preferably, liposomes, in the practice of thepresent disclosure, include small unilamellar liposome (SUV), largeunilamellar liposome (LUV), i.e., a unilamellar liposome with a diameterof greater than 50 nm and multilamellar liposomes (MLVs) having morethan one lipid bilayer.

In general, liposomes are composed of a lipid mixture including one ormore lipids. Examples of lipids includes, but not limited to, (i)neutral lipid, e.g. cholesterol, ceramide, diacylglycerol, acyl(polyethers) or alkylpoly(ethers); (ii) neutral phospholipid, e.g.,diacylphosphatidylcholines, sphingomyelins, anddiacylphosphatidylethanolamines, (iii) anionic lipid, e.g.,diacylphophatidylserine, diacylphosphatidylglycrol, diacylphosphatidate,cardiolipin, dacylphophatidylinositol, diacylglycerolhemisuccinate,diacylglycerolhemiglutarate, and the like; and (v) cationic lipid, e.g.,dimethyldioctadecylammonium bormide (DDAB), 1,2,-diacyl-3-trimethylammonium propane (DOTAP), and1,2-diacyl-sn-glycero-3-ethylphosphocholine.

In a typical case of the hydrophilic polymer derivatized liposome, theliposome is composed of a mixture of at least one phospholipid and aneutral lipid, and a hydrophilic polymer conjugated lipid. Examples ofthe phospholipid used in the present disclosure include, but are notlimited to, phosphatidylcholine (PC), phosphatidylglycerol (PG),phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidicacid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC), eggphosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), eggphosphatidylserine (EPS), egg phosphatidic acid (EPA), eggphosphatidylinositol (EPI), soy phosphatidylcholine (SPC), soyphosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soyphosphatidylserine (SPS), soy phosphatidic acid (SPA), soyphosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC),dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylglycerol (DOPG),dimyristoylphosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC),hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), di stearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylethanolamine (DOPE),palmitoylstearoylphosphatidylcholine (PSPC),palmitoylstearoylphosphatidylglycerol (PSPG),monooleoylphosphatidylethanolamine (MOPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylserine(DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS),dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine(DSPS), dipalmitoylphosphatidic acid (DPPA),1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA),dimyristoylphosphatidic acid (DMPA), di stearoylphosphatidic acid(DSPA), dipalmitoylphosphatidylinositol (DPPI),1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI),dimyristoylphosphatidylinositol (DMPI), distearoylphosphatidylinositol(DSPI), and a mixture thereof.

Hydrophilic Polymer Conjugated Lipid

The term “hydrophilic polymer conjugated lipid” refers to hydrophilicpolymer with a long chain of highly hydrated flexible neutral polymerattached to a lipid molecule. Examples of the hydrophilic polymerincludes, but not limited to, polyethylene glycol (PEG) with a molecularweight about 2,000 to about 5,000 daltons, methoxy PEG (mPEG),ganglioside GM₁, polysialic acid, polyglycolic acid,apolyacticpolyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone,polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline,polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,polyvinylmethylether, polyhydroxyethyl acrylate, derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose and syntheticpolymers. Examples of the lipid molecule includes, but not limited to(i) neutral lipid, e.g. cholesterol, ceramide, diacylglycerol, acyl(polyethers) or alkylpoly(ethers); (ii) neutral phospholipid, e.g.,diacylphosphatidylcholines, sphingomyelins, anddiacylphosphatidylethanolamines, (iii) anionic lipid, e.g.,diacylphophatidylserine, diacylphosphatidylglycrol, diacylphosphatidate,cardiolipin, dacylphophatidylinositol, diacylglycerolhemisuccinate,diacylglycerolhemiglutarate, and the like; and (v) cationic lipid, e.g.,dimethyldioctadecylammonium bormide (DDAB), 1,2,-diacyl-3-trimethylammonium propane (DOTAP), and1,2-diacyl-sn-glycero-3-ethylphosphocholine.

In one group of embodiment, the hydrophilic polymer conjugated lipid isthe polyethylene glycol-derived lipid, which includes, but not limitedto: DSPE-PEG, wherein the molecular weight of PEG is about 2,000 daltons(hereafter DSPE-PEG₂₀₀₀).

Encapsulation

The term “encapsulated” or “entrapped” compound, substance or atherapeutic agent refers to a compound, substance or a therapeutic agentis associated with the liposome or sequestered, at least in part, in theinternal compartment of liposome. The term “encapsulation efficiency”refers to the ratio of an amount of the liposomal form of drug to a sumof free form drug and liposomal form of drug. In one group ofembodiments, the encapsulation efficiency is calculated based on theamount of the entity encapsulated in the liposome divided by a sum of anamount of the entity not encapsulated in the liposome and the entityencapsulated in the liposome, which is determined using various methodsas known in the art.

Weak Acid Salt

The term “weak acid salt” refers to the conjugate base of the weak acid.As used herein, weak acid salt refers to both the conjugate base of theweak acid and to any accompanying counterion. Preferably, a weak acidsalt for use in the present disclosure is water soluble at highconcentrations. The counterion or cation should be practicallylipid-membrane impermeable (having permeability coefficient, P, of lessthan about 10⁻¹² to 10⁻¹¹ cm/s). The counterion may be monovalent ormultivalent. Exemplary weak acid for use in the present disclosureinclude carboxylic acids such as formic acid, acetic acid, propanoicacid, butanoic acid, pentanoic acid, and substituted derivativesthereof. Exemplary cation for use in the present disclosure includesodium, potassium, ammonium and calcium. Preferably, calcium is used ascation while prostaglandin is selected as the mild acidic agent inaccordance with the present disclosure.

Polyprotic Acid

The term “polyprotic acid” refers to weak organic polybasic acid.Exemplary polyprotic acid for use in the present disclosure includecitric acid, tartaric acid, succinic acid, adipic acid and aconiticacid, typically at 1 to 20 mM concentration.

Preferably, a polyprotic acid for use in the present disclosure iscitric acid at a concentration less than 3 mM; more preferably, lessthan 2.5 mM; and most preferably less than 2.3 mM. In a particularembodiment, the concentration of citric acid is from 1.5 to 2.5 mM.

Mild Acidic Agent

The term “mild acidic agent” as used herein refers to a compoundintended for loading into liposomes, and which also contains at leastone carboxy group and is amphipathic. The pKa of the agent is typicallyless than about 5. In a particular embodiment, the pKa of the agent isabout 4.85. The agent may also contain one or more functional groups inaddition to the carboxy function, although the presence of suchfunctional group should not significantly alter the acidity of the agentfrom that of its non-functionalized counterpart. The agent refers toboth the compound in its protonated form to any salt forms thereof. Asalt of the agent may be accompanied by any pharmaceutically acceptablecounterion.

The term “amphipathic” is used herein to denote a compound containingboth polar and nonpolar domains and thus having the ability to permeatenormally nonpermeable membrane under suitable conditions.

The term “amphipathic” is used herein to denote a molecule having bothhydrophobic (nonpolar) and hydrophilic (polar) groups, and beingcharacterized by any one of the following: pKa: it has a pKa above 3.0,preferably above 3.5, more preferably, in the range between about 3.5and about 6.5; Partition coefficient: in an n-octanol/buffer (aqueousphase) system having a pH of 7.0, it has a log D in the range betweenabout −3 and about 2.5.

Lyophilized Cake

The term “lyophilized cake” refers to a freeze-drying product containinga mild acidic agent, which prosssses desirable characteristics includingmaintenance of the characteristics of the original dosage form uponreconstitution, including solution properties; and particle-sizedistribution of suspensions; and isotonicity upon reconstitution.

II. Preparation of Liposome Solution for Loading

The present disclosure provides a liposome solution having liposomeswith a gradient of weak acid salt for loading a mild acidic agent acrossthe gradient. General methods for preparing the liposome solution isdescribed as below.

A. Liposome Formation

Liposomes with a weak acid salt entrapped within an aqueous interiorspace, which is suitable for forming the liposome solution in accordancewith the present disclosure, may be prepared by a variety of techniques.Examples of methods suitable for making liposomes of the presentdisclosure include solvent injection, reverse phase evaporation,sonication, microfluidisation, detergent dialysis, ether injection, anddehydration/rehydration. In a typical procedure, a lipid mixture isdissolved in ethanol and injected into a hydration buffer containing aweak acid salt, such as sodium or calcium acetate.

Typically, the membrane of the liposome is composed of a lipid mixture,wherein the lipid mixture includes a phospholipid or a mixture of atleast one phospholipid and neutral lipid, and a hydrophilic polymerconjugated lipid. In a preferable embodiment, the lipid mixture iscomposed of one or more phospholipid and cholesterol and a hydrophilicpolymer conjugated lipid, wherein the hydrophilic polymer conjugatedlipid is DSPE-mPEG at a molar percentage of less than 3% based on thetotal amount of the lipid mixture. In a group of embodiments, the molarpercentage of cholesterol ranges from 10% to 40% based on the totalamount of the lipid mixture. In one group of embodiments, thephospholipid is selected from DSPC, DMPC, DPPC and DOPC. In anothergroup of embodiments, the amount of phospholipid, cholesterol andDSPE-mPEG is at a molar ratio of 3:2:0.045.

The hydration buffer suitable for the present disclosure contains sodiumacetate, potassium acetate or calcium acetate. In one group ofembodiments, the hydration buffer contains sodium acetate and ispreferably at a concentration of at least 100 mM; and typically, between100 mM and 800 mM; preferably between 200 mM and 800 mM; and mostpreferably between 400 mM and 800 mM. In another group of embodiments,the hydration buffer contains calcium acetate at a concentrationpreferably between 200 mM and 400 mM; and more preferably 250 mM and 350mM. The hydration buffer is adjusted by addition of acid or base at a pHbetween 4.0 and 9.0, preferably between 7.5 and 8.5, and most preferablyof 8.2. In an alternative embodiment, while the hydration buffer furthercontains a polyprotic acid, such as citric acid, the preferable pH isadjusted to 6.5 and its retained PGE₁ could be relatively maintainedafter in vitro release assay in comparison to that without thepolyprotic acid.

For the mild acidic agent as well as the weak acid being allowed todistribute between inner and outer compartments, acting as an inside tooutside proton shuttle, retention of the mild acidic agent may alteramong a variety of components in the interior space of the liposome. Tomodulate the retention of the mild acidic agent, the hydration buffer isadjusted to contain a polyprotic acid. However, addition of thepolyprotic acid into the hydration buffer decrease the pH of the aqueousinterior space of the liposomes causes an undesirably reduction in theencapsulation efficiency. In a preferable embodiment, the hydrationbuffer contains a polyprotic acid at a concentration less than 50 mM,preferably less than 10 mM, more preferably between 0.01 mM and 5 mM,much more preferably between 0.1 mM and 3 mM, and most preferablybetween 1.5 mM and 2.5 mM.

The size of the liposomes can be controlled by controlling the pore sizeof membranes used for low pressure extrusion or the pressure and numberof passes utilized in microfluidisation or any other suitable method.The liposomes in accordance with the present disclosure have a meandiameter of about 30 nm to about 200 nm, more preferably about 50 nm toabout 150 nm.

B. Establishing Gradient of Weak Acid Salt

After sizing, the exterior hydration buffer of the liposomes isprocessed to form a higher inside concentration gradient of weak acidsalt, e.q. sodium acetate. This could be done by a variety oftechniques, e.g., by (a) dilution of an aqueous medium, (b) dialysisagainst an aqueous medium, (c) molecular sieve chromatography, or (d)high-speed centrifugation and resuspending of centrifuged liposomes inan aqueous medium, wherein the aqueous medium is suitable for physicalcondition and pharmaceutical administration.

In a general case, the aqueous medium, alternatively used as exteriorbuffer, contains a buffer and a solute for maintaining a desiredosmolarity and is adjusted pH at a range between 4 and 7, preferablybetween 4 and 5, and most preferably at 5.5. The exemplary buffer ishistidine, MES or the like at a pH 4 to 7 at a concentration at a rangeof 5 to 50 mM, and preferably 20 mM. The exemplary solute is salt of astrong acid, to form a saline, or mono- or di-sacchride, such assucrose, glucose, mannitol, lactose or maltose.

III. Preparation of Lyophilized Cake

Lyophilized cake for use in the present disclosure may be prepared by avariety of techniques. Typically, the lyophilization process consists ofthree stages: freezing, primary drying, and secondary drying. The designof a lyophilized formulation is dependent on the requirements of theactive pharmaceutical ingredient (API), herein the mild acidic agent,and intended route of administration. In an alternative embodiment, astock solution containing the mild acidic agent is applicable to thepresent disclosure and consists of: an organic solvent, such astert-Butyl alcohol (TBA); a cryoprotectant; and the mild acidic agent.The stock solution is subjected to the lyophilization process to obtainsaid lyophilized cake. The exemplary lyophilized cake contains a watercontent at a range of 1.2% to 1.5% and amounts of the mild acidic agentto cryoprotectant at a ratio of 0.0001:1.0 to 0.01:1.5, preferably,0.005:1.25, and more preferably, 0.0526:125 by weight. In a particularembodiment, the cryoprotectant is maltose.

IV. Method for Preparing Liposomal Composition Containing a Mild AcidicAgent

The hydrophilic polymer-derived liposomes encapsulating the mild acidicagent are prepared by the method according to the present disclosure.

In a typical procedure, a liposomal composition is prepared bycontacting a liposome solution in accordance with the present disclosurewith a lyophilized cake containing a mild acidic agent at a conditionfor the mild acidic agent to become encapsulated within the liposome,wherein the condition includes being at an ambient temperature,typically 18° C. to 30° C., for a period of time shorter than 20minutes. The liposome containing the mild acidic agent which isencapsulated within the aqueous interior space separated from theaqueous medium by the membrane is thus obtained and ready for use inpharmaceutical or other application.

In general, the liposomal composition is obtained by contacting theliposome solution and the lyophilized cake at a predetermined drug tolipid ratio. The drug to lipid ratio hereby refers to ratio of theamount of the mild acidic agent in the lyophilized cake to the amount oflipids in the liposome solution. An exemplary molar ratio of drug tolipid is from 0.001:1 to 0.05:1 Typically, the ratio of PGE₁:lipidranges from 20 μg:5 μmol to 10 μg:20 μmol.

In a group of embodiments, the condition includes, but not limited to atime sufficient for the mild acidic agent to become encapsulated withinthe liposome at an ambient temperature, the time is a least 5 minutes,for examples, 10 minutes, 60 minutes or 24 hours; and preferably atleast 10 minutes. More preferably, the condition includes allowing themild acidic agent to become encapsulated with the liposome at an ambienttemperature, which generally ranges from 18° C. to 30° C.

The resulting liposomal composition has encapsulation efficiency atleast 85%; and optionally at least 90%, 95%, or 97%; and preferably 95to 99%.

The disclosure will now be described in further detail with reference tothe following specific, non-limiting examples.

Example 1 Process for Preparing Lyophilized Cake

Cryoprotectant was dissolved in hot water (50˜60° C.) and then cooled tobelow 40° C. to form a cryoprotectant solution. Prostaglandin E₁ wasdissolved in tert-Butyl alcohol (TBA) being pre-warmed to liquefy thesolvent at 40° C. and then transferred to and mixed with thecryoprotectant solution to form a prostaglandin solution. Theprostaglandin solution was subjected to aseptic filtration and filledinto 6R vial, followed by lyophilization to form a lyophilized cakecontaining PGE₁ (hereby also denoted as alprostadil cake). Thelyophilization was performed with a shelf lyophilizer under a programwith the parameters as listed in the below Table I.

TABLE I The lyophilization parameters for alprostadil cake FreezePrimary drying Secondary drying End temperature (° C.) −40 −40 −30 30 4duration (min) 90 240 3000 720 — pressure (mTorr) — — 100 100 100

Example 2 Preparation of Alprostadil Cake with Various Components

To find a suitable formulation for forming the alprostadil cakeaccording to the present disclosure, 52.6 μg PGE₁ per vial was proposedfor alprostadil cake, 125 mg cryoprotectant was used for ourformulation.

As PGE₁ was used as API in the formulation, a suitable solvent wasneeded to dissolve water-insoluble PGE₁. PGE₁ is soluble in tert-Butylalcohol (TBA). TBA was selected to dissolve PGE₁ to form theprostaglandin solution.

PGE₁ undergo dehydration to form prostaglandin A₁. In order to increasethe shelf life of PGE₁, lyophilization was used to reduce the moisturecontent to a very low level to minimize the degradation. Screening ofthe candidate formulations were based on results of 40° C. acceleratedstability test.

As the content of cryoprotectant and PGE₁ per vial was fixed as above,the other lyophilization parameters, such as water content, TBA additionor others were further modulated as described in Table II andinvestigated as described in the following examples.

TABLE II Formulation of the alprostadil cake Formu- Type of Compositionper vial: mg (% (w:w)) lation cryo- cryo- Code protectant PGE₁* TBAprotectant water Pc029 lactose 0.0526 13.8 125 1211.1 monohydrate(0.0039) (1.02) (9.26) (89.71) Pc030 lactose 0.0526 10.8 125 914.2monohydrate (0.0050) (1.02) (11.90) (87.07) Pc031 lactose 0.0526 9.2 125765.7 monohydrate (0.0058) (1.02) (13.89) (85.08) Pc032 lactose 0.05267.7 125 617.3 monohydrate (0.0070) (1.02) (16.67) (82.30) Pc033 lactose0.0526 16.5 125 608.5 monohydrate (0.0070) (2.19) (16.67) (81.13) Pc034lactose 0.0526 27.4 125 597.5 monohydrate (0.0070) (3.66) (16.67)(79.67) Pc035 sucrose 0.0526 7.7 125 617.3 (0.0070) (1.02) (16.67)(82.30) Pc036 maltose 0.0526 7.7 125 617.3 monohydrate (0.0070) (1.02)(16.67) (82.30) * 52.6 μg instead of 50 μg of PGE₁ was used hereby toadjust the final concentration of PGE₁ after reconstitution by 5 mLliposome solution to be 10 μg/mL.

Example 2A Effect of Water Content in the Prostaglandin Solution

First, upon the fixed percentage of TBA, the effect of percentage ofwater before lyophilization was investigated by using the formulationsas listed in Table III to form the alprostadil cakes.

TABLE III Formulations of the prostaglandin solutions with various watercontent Composition per vial: mg (% (w:w)) Formulation lactose CodePGE₁* TBA monohydrate water Pc029 0.0526 13.8 (1.02) 125 (9.26)  1211.1(89.71) (0.0039) Pc030 0.0526 10.8 (1.02) 125 (11.90)  914.2 (87.07)(0.0050) Pc031 0.0526  9.2 (1.02) 125 (13.89)  765.7 (85.08) (0.0058)Pc032 0.0526  7.7 (1.02) 125 (16.67)  617.3 (82.30) (0.0070) *52.6 μginstead of 50 μg of PGE₁ was used hereby to adjust the finalconcentration of PGE₁ after reconstitution by 5 mL liposome solution tobe 10 μg/mL

As shown in Table IV, all formulations met the criteria of 90% PGE₁remaining after 6 weeks at 40° C., the remaining content decreased aspercentage of water in the prostaglandin solution decreased. Itdemonstrated that reduction of the opportunity of contact of API withwater by increasing the inter-space (by increasing the percentage ofwater) could improve the stability of alprostadil cakes. Furthermore, nosignificant differences between Formulations Pc029 and Pc030demonstrated that as inter-space increased to a certain value, API andwater was separated at a large enough space. In order to minimize thetime of lyophilization, the formulation with the least water content wasthe favorable candidate. Formulation Pc030 was selected instead ofFormulation Pc029.

TABLE IV Stability of the obtained alprostadil cakes at 40° C. after 6weeks Formulation remaining PGE₁ Code (%) PGA₁ (%) water content (%)Pc029 95.9 ± 0.5 4.0 ± 0.4 1.40 ± 0.15 Pc030 96.1 ± 0.5 4.3 ± 0.0 1.34 ±0.03 Pc031 93.6 ± 1.0  5.2 ± 0.12 1.41 ± 0.04

Example 2B Effect of Solvent Content in the Prostaglandin Solution

Second, effect of the content of TBA in the PGE₁ solution wasinvestigated by using the formulations as listed in Table V to form thealprostadil cakes.

TABLE V Formulations of the prostaglandin solutions with various TBAconcentrations Composition per vial: mg (% (w:w)) Formulation lactoseCode PGE₁ TBA monohydrate water Pc032 0.0526 7.7 125 (16.67) 617.3(82.30) (0.0070) (1.02) Pc033 0.0526 16.5 (2.19) 125 (16.67) 608.5(81.13) (0.0070) Pc034 0.0526 27.4 125 (16.67) 597.5 (79.67) (0.0070)(3.66)

As shown in Table VI, increase of the TBA content slowed the degradationof PGE₁. It was suggested to be caused from variance of crystallizationin freeze step, a loose apparent cake was observed in the group of highTBA content in comparison to a crystal-like in the group of low TBAcontent, which indicated crystallization varied in the groups withvarious TBA content. The variance of crystal conditions influencedstability of PGE₁ and led to the observed results.

TABLE VI Stability of the obtained alprostadil cakes at 40° C. after 6weeks Formulation remaining PGE₁ number (%) PGA₁ (%) water content (%)Pc032 90.7 ± 1.0 9.7 ± 1.1 1.27 ± 0.02 Pc033 95.2 ± 1.1 3.6 ± 0.4 1.35 ±0.07 Pc034 94.9 ± 2.5 5.0 ± 0.8 1.36 ± 0.02

Example 2C Effect of Cryoprotectant Content in the ProstaglandinSolution

Finally, effect of the different cryoprotectants as identified in TableVII were also investigated by using formulations as listed in Table VIIto from the alprostadil cakes.

TABLE VII Formulations of the prostaglandin solutions with variouscryoprotectants Formu- Composition per vial: mg (% (w:w)) lation cryo-cryo- Code protectant PGE₁ TBA protectant water Pc032 lactose 0.0526 7.7125 617.3 monohydrate (0.0070) (1.02) (16.67) (82.30) Pc035 sucrose0.0526 7.7 125 617.3 (0.0070) (1.02) (16.67) (82.30) Pc036 maltose0.0526 7.7 125 617.3 monohydrate (0.0070) (1.02) (16.67) (82.30)

As shown in Table VIII, the alprostadil cake with maltose ascryoprotectant showed the slowest degradation rate (Pc036). On the otherhand, the alprostadil cake with sucrose as cryoprotectant (Pc035) wasthe worst among all formulations. This indicated species ofcryoprotectant could modulate the stability of PGE₁ in the alprostadilcake, resulting from variance of crystal structure among allformulations during lyophilization.

TABLE VIII Stability of the obtained alprostadil cakes at 40° C. after 6weeks Formulation remaining PGA₁ water Code PGE₁ (%) (%) content (%)Pc032 90.7 ± 1.0^(a)  9.7 ± 1.1^(a) 1.27 ± 0.02^(a) Pc035 87.7 ± 0.9^(b)10.4 ± 0.8^(b) 1.17 ± 0.05^(b) Pc036 93.9 ± 0.4^(b)  6.5 ± 0.3^(b) 1.16± 0.01^(b)

Results

Based on the results above, higher water content, a higher TBA contentand maltose used as cryoprotectant in the prostaglandin solution wereselected to improve the stability of cake.

The stability of Pc030 was further evaluated based on the result of 40°C. accelerated stability test and only 4% degradation was observed after6 weeks incubation. Formulation Pc030 was selected for its slowestdegradation rate of PGE₁ with lactose monohydrate. Lactose monohydratewas selected as the cryoprotectant in the following examples.Accordingly, Pc030 was selected as a suitable formulation foralprostadil cake.

TABLE IX Candidate formulation of the alprostadil cake Composition pervial: mg (% (w:w)) Formulation lactose number PGE₁* TBA monohydratewater Pc030 0.0526 10.8 125 914.2 (0.0050) (1.02) (11.90) (87.07)

Example 3 General Process for Preparing Liposome Solution

Liposome was composted with HSPC, cholesterol, and optional1,2-Distearoyl-phosphatidyl ethanolamine-methyl-polyethyleneglycolconjugate (DSPE-mPEG). For forming liposome particles, the lipids weremixed and dissolved in ethanol first; and then injected into anhydration buffer, wherein the hydration buffer contains a weak acidsalt, such as sodium acetate, at a pH value higher than 7. Due toamphipathic properties of lipid molecule, the liposome particles werespontaneously formed and suspended in the hydration buffer.

After liposome particles forming, the particle size was manipulated byextrusion, which liposome particles are forced to pass thoroughpolycarbonate membrane to obtain liposomes with an average diameter at arange of 80 nm to 180 nm. After multiple passing, the liposome particlesize was down to sub-micrometer level; and the liposome particles weremore stable then initial particles.

The exterior hydration buffer which liposomes suspending in werereplaced by diafiltration using tangent flow filtration (TFF) with anexterior buffer to form an aqueous medium suspending the liposomes;also, some of acetate molecule inside liposomes could penetrate liposomebilayer and be removed during this process. This could generate anacetate/pH gradient across the liposome membrane; and such gradient wasessential for PGE₁ active loading (i.e. remote loading).

Accordingly, the liposome solution for PGE₁ remote loading was prepared.The following experiments were designed to investigate suitableformulation parameters, process parameters, and PGE₁ remote loadingparameters to achieve high encapsulation efficiency and sustainedrelease of PGE₁.

Encapsulation efficiency was determined by a general process comprisingthe following steps. Liposomal and free form PGE₁ were separated byself-packing resin column: Firstly, a column with Toyopearl HW-55F resinin a Bio-Rad column was prepared. The sample was applied into theconditioned column and eluted by normal saline for collecting fractionsof liposomal PGE₁, followed by eluting the column with water forcollecting fractions of free form PGE₁. Both contents of liposomal andfree form PGE₁ were analyzed by HPLC. The encapsulation efficiency wascalculated as dividing the amount of the liposomal form PGE₁ with thecombined amounts of the liposomal and free form PGE₁.

In vitro release assay was performed by a general process comprising thefollowing steps. PGE₁-loaded liposome was mixed with sodium acetatebuffer or human plasma (Example 6A) for triggering and acceleratingrelease of PGE₁ from the liposome. The encapsulation efficiency afterthe release assay was determined according to the above description asthe retained PGE₁.

Example 4 Preparation of Liposome Solution

The liposome solutions were prepared according to the process asdescribed in Example 3, including the descriptions of the lipid, thehydration buffer, the aqueous medium and the other parameters in theprocedures of PGE₁ loading, such as a step of contacting the liposomesolutions with lyophilized cakes, e.g., alprostadil cakes as previouslydescribed, to conduct or alprostadil in a form of solution, e.g. PGE₁solution; and forming liposomal compositions. Other parameters includingloading durations, drug-to-lipid ratio and final obtained encapsulationefficiency are shown in Table X.

TABLE X Formulations of the liposome solution Formulation Lipidhydration buffer/ aqueous Code composition pH medium PL111DSPC:cholesterol:DSPE- 400 mM NaAc/6.5 Double distilled mPEG = 3:2:0.045water(ddH₂O) PL112 DSPC:cholesterol:DSPE- 400 mM NaAc/8.2 ddH₂O mPEG =3:2:0.045 PL114 DSPC:cholesterol:DSPE- 400 mM NaAc/4.0 ddH₂O mPEG =3:2:0.045 PL115 DSPC:cholesterol:DSPE- 200 mM Ca(Ac)₂/7.6 ddH₂O mPEG =3:2:0.045 PL116 DSPC:cholesterol:DSPE- 400 mM NH₄Ac/6.9 ddH₂O mPEG =3:2:0.045 PL117 DSPC:cholesterol:DSPE- 400 mM KAc/7.8 ddH₂O mPEG =3:2:0.045 PL130 DSPC:cholesterol:DSPE- 400 mM NaAc/8.2 20 mM histidinein 6% mPEG = 3:2:0.045 sucrose, pH 6.0 PL141 DSPC:cholesterol:DSPE- 100mM NaAc/8.1 20 mM histidine in 6% mPEG = 3:2:0.045 sucrose, pH 5.5 PL142DSPC:cholesterol:DSPE- 200 mM NaAc/8.3 20 mM histidine in 6% mPEG =3:2:0.045 sucrose, pH 5.5 PL143 DSPC:cholesterol:DSPE- 600 mM NaAc/8.820 mM histidine in 6% mPEG = 3:2:0.045 sucrose, pH 5.5 PL144DSPC:cholesterol:DSPE- 800 mM NaAc/8.9 20 mM histidine in 6% mPEG =3:2:0.045 sucrose, pH 5.5 PL153 DSPC:cholesterol:DSPE- 400 mM NaAc + 20mM histidine in 6% mPEG = 3:2:0.045 50 mM citric acid/5.07 sucrose, pH5.5 PL154 DSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 6%mPEG = 3:2:0.045 100 mM citric acid/4.70 sucrose, pH 5.5 PL155DSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 6% mPEG =3:2:0.045 200 mM citric acid/4.19 sucrose, pH 5.5 PL156DSPC:cholesterol:DSPE- 400 mM NaAc/8.2 20 mM histidine in 6% mPEG =3:2:0.045 sucrose, pH 5.5 PL157 DSPC:cholesterol:DSPE- 400 mM NaAc/8.220 mM histidine in 6% mPEG = 3:2:0.045 sucrose, pH 5.5 PL158DSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 6% mPEG =3:2:0.045 citric acid/6.5 sucrose, pH 5.5 PL162 DSPC:cholesterol:DSPE-400 mM NaAc/8.2 20 mM histidine in 6% mPEG = 3:2:0.045 sucrose, pH 5.5PL177 HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 6% mPEG =3:2:0.045 2 mM citric acid/6.5 sucrose, pH 5.5 PL178HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 6% mPEG =3:2:0.045 2 mM citric acid/6.5 sucrose, pH 5.5 PL194HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG =3:2:0.045 2 mM citric acid/6.5 lactose monohydrat, pH 5.5 PL196HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG = 3:2:02 mM citric acid/6.5 lactose monohydrat, pH 5.5 PL197HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG =3:2:0.02 2 mM citric acid/6.5 lactose monohydrat, pH 5.5 PL198HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG =3:2:0.15 2 mM citric acid/6.5 lactose monohydrat, pH 5.5 PL199HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG =3:2:0.32 2 mM citric acid/6.5 lactose monohydrat, pH 5.5 PL200HSPC:cholesterol:DSPE- 400 mM NaAc + 20 mM histidine in 7% mPEG =3:2:0.56 2 mM citric acid/6.5 lactose monohydrat, pH 5.5 NaAc representssodium acetate; Ca(Ac)₂ represents calcium acetate; NH₄Ac representsammonium acetate; and KAc represents potassium acetate.

Example 5 Preparation of Liposome Solution Under Various Conditions forImproving Encapsulation Efficiency

Higher encapsulation efficiency represents minimization of free formPGE₁. Improving encapsulation efficiency of PGE₁ after PGE₁ loading inthe finally obtained liposomal composition was prioritized. The exteriorand interior phase at both sides of liposome membrane were modified bychanging hydration buffers and aqueous mediums to form an increaseddriving force for PGE₁ loading in this Example.

Example 5A pH Modification of the Hydration Buffer

pH value of the hydration buffer determined interior pH of liposomes.Though the interior pH could be elevated after diafiltration process byinterior acetate penetrating out, the pH of the hydration bufferremained majorly contribute to the gradient across the liposome membranefor PGE₁ loading.

The influence of interior pH by the hydration buffer containing acetateon encapsulation efficiency was evaluated and summarized in Table XI.

TABLE XI Formulations with the hydration buffer at various pH valueshydration encapsulation Formulation buffer efficiency Code Buffer pH (%)PL114 NaAc 4.0 74.1 PL111 NaAc 6.5 70.0 PL112 NaAc 8.2 82.9Encapsulation efficiency was assayed under identical loading conditionof a drug-to-lipid ratio 20 μg:10 mol, and 60° C. incubation for 5minutes.

The result shows that higher buffer pH had better encapsulationefficiency. This result correlated remote loading theory: greater pHgradient with better encapsulation efficiency. The pH value of thehydration buffer was determined to be 8.2.

Example 5B pH Modification of the Aqueous Medium

pH value of the aqueous medium determined exterior pH of liposomes andalso contributed to PGE₁ loading by influencing pH gradient across theliposome membrane. The influence of pH of the aqueous medium containinghistidine at a concentration of 20 mM by adjusting pH to the levels isindicated in Table XII.

TABLE XII pH optimization of the aqueous medium Formulation pH of theaqueous encapsulation Code medium efficiency (%) PL130 4 92.1 5 91.8 5.588.4 6 89.1 6.5 64.9 7 69.1

Encapsulation efficiency was assayed under the following loadingcondition of a drug-to-lipid ratio 10 μg:5 μmol, and 20° C. incubationfor 30 minutes.

Since the pH of the aqueous medium determines pH gradient across theliposome membrane, exterior pH higher than 6.0 resulted to dramaticallydecrease of encapsulation efficiency. The pH value of the aqueous mediumwas determined to be 5.5.

Example 5C Concentration Modification of Hydration Buffer

Concentration of the weak acid salt in the hydration buffer was anotherfactor which might influence the encapsulation efficiency.

The influence of the concentrations of the weak acid salt as indicatedin Table XIII on E.E. was evaluated and summarized in Table XIII.

TABLE XIII Hydration buffer concentration concentration of NaAc inencapsulation Formulation the hydration efficiency Code buffer (mM) (%)PL141 100 68.1 PL142 200 78.0 PL130 400 86.2 PL143 600 86.5 PL144 80087.0

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 10 μg:5 μmol, and 25° C. incubation for30 minutes.

E.E. reached plateau when concentration of sodium acetate was more than400 mM. Higher acetate concentration did not alter encapsulationefficiency. However, a higher acetate concentration remained essentialfor forming acetate gradient for PGE₁ loading since low acetateconcentration showed lower E.E.

The concentration of the weak acid salt was determined to be 400 mMsodium acetate while it generated enough gradient for PGE₁ loading.

Results

According to Examples 5A to 5C, the properties of the hydration bufferand the aqueous medium were determined using encapsulation approach. Thehydration buffer containing 400 mM sodium acetate at pH 8.2 and theaqueous medium containing 20 mM histidine at pH 5.5 showed a desiredencapsulation efficiency.

Example 6 Preparation of Liposome Solution Under Various Conditions forImproving Sustained Release

A target liposomal composition according to the present disclosurerequired a sustained release property to control drug release in humanbody. Thus, the following modifications were evaluated by not onlyencapsulation efficiency but also in vitro release assay to select adesired formulation.

The retained PGE percentage was determined by the method as described inthe previous example.

Example 6A Modification of Cation in the Hydration Buffer

Cation of the weak acid salt in the hydration buffer interacted withPGE₁ as an anion after PGE₁ loading. Such interaction might slow downPGE₁ release. Different acetate salts were used as the weak acid saltsin the hydration buffers and evaluated by encapsulation efficiency assayand in vitro release assay with human plasma.

TABLE XIV Various cations used in the hydration buffers Formulationhydration encapsulation retained PGE₁ Code buffer efficiency (%)percentage (%)* PL112 400 mM NaAc 87.6 13.0 PL115 200 mM Ca(Ac)₂ 91.10.0 PL116 400 mM NH₄Ac 62.0 12.2 PL117 400 mM KAc 79.6 34.1 *Usingpurified liposome form fraction for in-vitro release study.

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 20 μg:10 umol, 60° C. incubation for 5minutes. In vitro release was conducted by mixing an analyte with humanplasma at a ratio being 1:1, and then incubated at 37° C. for 2 hours.

Only sodium acetate and potassium acetate liposome represented both highencapsulation efficiency and retained PGE₁ after plasma release. Furtherinvestigations focused on these two selected formulations as shown inTable XV.

TABLE XV in-vitro release results of Formulations PL112 and PL117Formulation hydration encapsulation retained PGE₁ Code buffer efficiency(%) percentage (%)* PL112 400 mM NaAc, 84.4 19.5 ± 1.8 pH 8.2 (n = 2)PL117 400 mM KAc, 73.2 28.8 ± 3.3 pH 7.8 (n = 2) *Using purifiedliposome form fraction for in-vitro release study.

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 20 μg:10 μmol, 60° C. incubation for 5min. In vitro release was performed by mixing an analyte with humanplasma at a ratio being 1:1, and then incubated at 37° C. for 1 hour.

Although potassium acetate showed a desired release profile in plasmarelease assay, its initial encapsulation efficiency was much less thanthe group with sodium acetate as the weak acid salt. Thus, sodiumacetate was selected as the weak acid salt in the hydration buffer.

Example 6B Addition of Citric Acid in the Hydration Buffer

According to Example 5, the hydration buffer was determined as 400 mMsodium acetate at pH 8.2. However, high pH might lead to instability oflipid. To avoid lipid instability and establishing greater acetategradient for loading, the influence of additional citric acid at variousconcentrations as indicated in Table XVI were evaluated.

TABLE XVI Hydration buffers with various citric acid concentrationsaddition of citric acid in hydration encapsulation Formulation hydrationbuffer (mM) buffer pH efficiency (%) PL130 0 8.4 87.6 PL153 50 5.1 83.3PL154 100 4.7 69.2 PL155 200 4.2 26.5

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 10 μg:5 umol, and 20° C. incubation for30 minutes.

It demonstrated that additional of citric acid decreased encapsulationefficiency. However, the decease of E.E. might result from the dramaticpH decrease which was against PGE₁ loading.

Thus, the effect of addition of citric acid to maintain a pH of 6.5 asshown in Table XVII was further evaluated by encapsulation efficiency(E.E.) and In vitro release (IVR).

TABLE XVII Modification of the hydration buffer with additional citricacid encapsulation E.E. Formulation hydration hydration efficiency afterCode buffer buffer pH (%) IVR (%) PL162 400 mM NaAc 8.2 87.4 25.1 ± 3.3(n = 3) PL158 400 mM 6.5 92.8 33.9 ± 2.0 NaAc + 1.5 mM (n = 3) citricacid

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 10 μg:5 umol, and 20° C. incubation for30 min. IVR was performed at a ratio of 1:1 to be mixed with 15 mM NaAcwith 8.1% sucrose buffer, pH 5.5, and then incubated at 25° C. for 30minutes.

According to the result, additional citric acid in the hydration buffernot only increased encapsulation efficiency, but also kept more PGE₁inside liposome after IVR. Accordingly, the aqueous medium was thusdetermined to be 400 mM of sodium acetate with citric acid at aconcentration ranging from 1.5 mM to 2 mM at pH 6.5.

Example 6C Preparation of Liposomal Composition by Modification ofDrug-to-Lipid Ratio

Liposome solutions prepared according to Table X were respectivelycontacted with a lyophilized cake containing PGE₁ or a PGE₁ solution ata drug-to-lipid ratio as indicated below in Table XVIII (a) to formcorresponding liposomal compositions. The PGE₁ solution was prepared bydissolving PGE₁ powder in an appropriate amount of ethanol to obtain aPGE₁ stock solution at a concentration of 5 mg/mL. For preparingliposomal composition according to the present disclosure, the liposomesolution and the PGE₁ stock solution were diluted into 9% sucrosesolution to a target concentration; and then incubated at an indicatedcondition for PGE₁ loading into liposomes.

TABLE XVIII (a) Drug-to-lipid ratio modification Formulation loadingloading duration drug-to-lipid ratio Code temperature (° C.) (min)(PGE₁:lipid) PL111 60° C. 5 min 20 μg: 10 μmol^(a) PL112 60° C. or 20°C. 5 min or 30 min 20 μg: 10 μmol^(a) PL114 60° C. 5 min 20 μg: 10μmol^(a) PL115 60° C. 5 min 20 μg: 10 μmol^(a) PL116 60° C. 5 min 20 μg:10 μmol^(a) PL117 60° C. 5 min 20 μg: 10 μmol^(a) PL130 20° C. or 25° C.10 min or 30 min 10 μg: 5 μmol^(b) PL141 25° C. 30 min 10 μg: 5 μmol^(b)PL142 25° C. 30 min 10 μg: 5 μmol^(b) PL143 25° C. 30 min 10 μg: 5μmol^(b) PL144 25° C. 30 min 10 μg: 5 μmol^(b) PL153 25° C. 30 min 10μg: 5 μmol^(b) PL154 25° C. 30 min 10 μg: 5 μmol^(b) PL155 25° C. 30 min10 μg: 5 μmol^(b) PL156 25° C. 30 min 10 μg: 10 μmol^(b) PL157 25° C. 30min 10 μg: 20 μmol^(b) PL158 25° C. 30 min 10 μg: 5 μmol^(b) PL162 25°C. 30 min 10 μg: 5 μmol^(b) PL177 25° C. 10 min 10 μg: 10 μmol^(b) PL17825° C. 10 min 10 μg: 20 μmol^(b) PL194 25° C. 10 min 10 μg: 10 μmol^(b)PL196 25° C. 10 min 10 μg: 10 μmol^(b) PL197 25° C. 10 min 10 μg: 10μmol^(b) PL198 25° C. 10 min 10 μg: 10 μmol^(b) PL199 25° C. 10 min 10μg: 10 μmol^(b) PL200 25° C. 10 min 10 μg: 10 μmol^(b) After PGE₁loading at a drug-to-lipid ratio ranging from 5 to 20 μg to 10 μmol at acondition of at a incubation temperature ranging from 20 to 60° C. for 5to 30 minutes, the encapsulation efficiency of PGE₁ in the obtainedliposomal composition by mixing the liposome solutions with thealprostadil cake were evaluated respectively, ranging from about 70% to97.1 ± 1.2%. ^(a)Liposome solution was loaded with a PGE₁ solution.^(b)Liposome solution was loaded with a lyophilized cake containingPGE₁.

In initial formulation, the drug-to-lipid ratio was predetermined to be10 μg:5 μmol. To further increase the encapsulation efficiency andsustain release property of liposomes, drug-to-lipid ratio in PGE₁loading was modified and results of E.E of PL130, PL156 and PL 157 weresummarized as in Table XVIII (b).

TABLE VIII (b) Drug-to-lipid ratio modification drug-to- encapsulationFormulation lipid ratio efficiency (%) PL130 10 μg: 5 μmol 87.6 PL156 10μg: 10 μmol 91.9 PL157 10 μg: 20 μmol 97.1 ± 1.2 (n = 4)

Encapsulation efficiency was assayed under the following loadingcondition: 25° C. incubation for 30 minutes.

Lower drug-to-lipid ratio increased encapsulation efficiency.Afterwards, further tested were the drug-to-lipid ratios at 10 μg:10μmol and 10 μg:20 μmol liposomes at a condition of incubation at 25° C.for 10 minutes.

TABLE XIX Lower drug-to-lipid ratio liposomes drug- encapsulation E.E.after to-lipid efficiency (%) IVR (%) Formulation ratio (n = 3) (n = 3)PL177 10 μg: 10 μmol 92.9 ± 1.7 36.62 ± 1.35 PL178 10 μg: 20 μmol 95.6 ±1.7 48.83 ± 3.02

Encapsulation efficiency was assayed under the following loadingcondition: 25° C. incubation for 10 minutes. IVR was performed at aratio of 1:1 to mix with 15 mM NaAc with 8.1% sucrose buffer, pH 5.5,and then incubated at 25° C. for 15 minutes.

Lowering drug-to-lipid ratio slightly increased both encapsulationefficiency and E.E. after IVR. However, drug-to-lipid ratio as 10 μg:20μmol was so low. Therefore, the drug to lipid ratio of 10 μg:10 μmol wasdetermined.

Example 7 Modulation of mPEG Content

The amounts of phosphocholine and cholesterol were fixed at a molarratio being 3:2 because this type of composition gives greatest condensearrangement of lipids. As for DSPE-mPEG, which generated surfacenegative charge for avoiding liposome clearance in human body orliposome aggregation, altered PGE₁ loading and IVR as shown in Table XX.

TABLE XX Modulation of mPEG content mPEG encapsulation E.E. aftercontent efficiency IVR Formulation (mol %) (%) (%) PL196 0 76.6 52.6PL197 0.4 83.5 49.9 PL194 0.9 98.6 59.1 PL198 3 95.5 58.6 PL199 6 85.436.9 PL200 10 86.7 40.9

Encapsulation efficiency was assayed under the following loadingcondition: drug-to-lipid ratio 10 μg:10 μmol, and 25° C. incubation for10 minutes. For IVR assay, after 1 hour of PGE₁ loading (for equilibriumloading), the obtained liposome solution was at a ratio 9:1 (v/v) mixedwith 75 mM sodium acetate releasing buffer, and incubated at 37° C. for15 minutes.

As shown in Table XX and FIG. 2, DSPE-mPEG molar ratio between 0.9% and3% (i.e. HSPC:cholesterol:DSPE-mPEG=3:2:0:045 to 3:2:0.15) shown highestencapsulation efficiency under incubation for 10 minutes. Further, theamount of the retained PGE₁ ranked high among these formulations afterIVR assay. The final lipid composition was determined to contain lipidat a ratio of HSPC:cholesterol:DSPE-mPEG=3:2:0:045.

Results

According to Examples 6A to 6C and 7, the formulation of liposomesolution was modified as a lipid composition withHSPC:cholesterol:DSPE-mPEG=3:2:0.045; the hydration buffer containing400 mM of sodium acetate and 2 mM of citric acid at pH 6.5; and thedrug-to-lipid ratio of 10 μg:10 μmol as shown in Table XXI.

TABLE XXI Composition of PL194 formulation Formulation lipid molar ratiohydration buffer aqueous medium code HSPC Cholesterol DSPE-mPEG Salt pHSalt pH PL194 3 2 0.045 400 mM 6.5 20 mM 5.5 NaAc + 2 histidine + mMcitric 7% Lactose acid monohydrate

Such formulation showed high PGE₁ encapsulation efficiency (about 90%)after loading at ambient temperature within a short period of time (10minutes); and retained more PGE₁ than other formulations in IVR assay.

Example 8 In Vivo Evaluation of Efficacy of PGE₁ Formulations by UsingBlood Flow Analysis

The efficacy of the liposomal compositions PL157 and PL194 was comparedto free-drug (PEG₁-CD) treated animals by blood flow study as describedbelow.

Blood Flow Study

Wistar rats were anesthetized by isoflurane and kept body temperature ona heating pad (36.5° C.). When the animals were deeply anesthetized,cutaneous microvascular blood flow of toruli digitales on the digitusquintus of right caudal paw was recorded by Laser Doppler Flowmetry(MoorVMS LDF1, Moor Instruments Ltd) 5 minutes before intravenousadministration of 3 ug/kg of PGE₁-CD or the liposomal compositions andthen continuously recorded for another 20 minutes to monitor theresponse after dose. Raw data of flux value was recorded by software ofmoorVNS-PCV2.0 and exported to Excel file (Microsoft software) for dataprocessing.

The blood flow data was normalized and plotted on graphs, and theparameters BF₀, BF_(max) and AUC % were calculated, wherein BF₀represents mean blood flow of baseline; BF_(max) represents maximumblood flow post dose; AUC % represents the percentage of area under thecurve from 0 to 20 min of blood flow compared to baseline AUC.

Results

As shown in Table XXII and Table XXIII, both PL194 and PL157 causedhigher BF_(max)/BF₀ and larger AUC ratio in comparison to PGE₁-CD.

PL194 caused slightly lower BF_(max)/BF₀ ratio and smaller AUC ratiocompared to PL157.

TABLE XXII Study 1 Dose No. of BF₀ BF_(max) Formulation (μg/kg) animal(mL/min/100 g) (mL/min/100 g) BF_(max)/BF₀ AUC % PL157 3 5MX3 10.5 ± 0.518.7 ± 1.7* 1.8 ± 0.2^(#) 142.4 ± 9.9^(#) PGE₁-CD 3 11.7 ± 0.6 15.7 ±0.8* 1.3 ± 0.0  117.2 ± 3.5  Mean ± S.E. *p < 0.05 as compared to BF₀;^(#)p < 0.05 as compared to PGE₁- CD

TABLE XXIII Study 2 Dose No. of BF₀ BF_(max) Formulation (μg/kg) animal(mL/min/100 g) (mL/min/100 g) BF_(max)/BF₀ AUC % PL194 3 7M 15.6 ± 1.425.2 ± 4.2* 1.6 ± 0.1^(#) 137.1 ± 8.2^(#) PGE₁-CD 3 16.3 ± 1.3 22.6 ±5.9* 1.4 ± 0.0  117.2 ± 2.0  Mean ± S.E. *p < 0.05 as compared to BF₀;^(#)p < 0.05 as compared to PGE₁- CD

The formulations of both the alprostadil cake and liposome solution wereinvestigated as above. The formulation processed desired propertiesreflected by stability test and animal study. For the alprostadil cake,Formulation Pc030 or Pc036, showed the preferable stability among theothers. For the liposome solution, Formulation PL194, showed highlyencapsulation efficiency within a short loading duration at ambienttemperature condition; also, the retained PGE₁ was more than otherformulations in IVR study.

As results of blood flow shown, the liposomal composition according tothe present disclosure indeed acquires an improved efficacy incomparison to the conventional prostaglandin composition.

Even though numerous characteristics and advantages of the presentdisclosure have been set forth in the foregoing description, togetherwith details of the structure and features of the disclosure, thedisclosure is illustrative only. Changes may be made in the details,especially in matters of shape, size, and arrangement of parts withinthe principles of the disclosure to the full extent indicated by thebroad general meaning of the terms in which the appended claims areexpressed.

This application claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application Nos. 62/372,096, filed Aug. 8, 2016 and62/375,698, filed Aug. 16, 2016, which applications are herebyincorporated by reference in their entirety.

1-5. (canceled)
 6. A delivery vehicle, said delivery vehicle comprisinga liposome in an aqueous medium, wherein the liposome comprises acombination of a weak acid salt and a polyprotic acid encapsulatedwithin the liposome.
 7. The delivery vehicle according to claim 6,wherein the polyprotic acid is an organic tribasic acid.
 8. The deliveryvehicle according to claim 6, wherein the polyprotic acid is selectedfrom the group consisting of citric acid, succinic acid, tartaric acidor a combination thereof. 9-33. (canceled)
 34. A liposomal composition,comprising a liposome-encapsuled mild acidic agent in an aqueous mediumand prepared by the steps: contacting the liposome of the deliveryvehicle according to claim 6 with a mild acidic agent to obtain theliposome-encapsulated mild acidic agent.
 35. The liposomal compositionaccording to claim 34, wherein the liposome comprises a lipid mixture;and the lipid mixture contains one or more lipids and a polyethyleneglycol-derived lipid.
 36. The liposomal composition according to claim34, wherein the polyethylene glycol-derived lipid is at a molarpercentage ranging from 0.1% to 3% based on the total amount of thelipid mixture; preferably from 0.5% to 3%; and more preferably, from0.5% to 1%.
 37. The liposomal composition according to claim 34, whereinthe weak acid salt is sodium acetate, potassium acetate or calciumacetate.
 38. The liposomal composition according to claim 34, whereinthe weak acid salt is at a concentration of at least 100 mM; andtypically, between 100 mM and 800 mM; preferably between 200 mM and 800mM; and most preferably between 400 mM and 800 mM. 39-41. (canceled) 42.The liposomal composition according to claim 34, wherein the polyproticacid is at a concentration less than 50 mM, preferably less than 10 mM,more preferably between 0.01 mM and 5 mM, much more preferably between0.1 mM and 3 mM, and most preferably between 1.5 mM and 2.5 mM.
 43. Theliposomal composition according to claim 34, wherein the molar ratio ofthe mild acidic agent to the amount of lipids of the liposomalcomposition is at least 0.001:1 and preferably from 0.001:1 to 0.05:1.44. The delivery vehicle according to claim 6, wherein the combinationof the weak acid salt and the polyprotic acid is at a molar ratio noless than 8:1.
 45. The delivery vehicle according to claim 6, whereinthe combination of the weak acid salt and the polyprotic acid is at amolar ratio ranging from 8:1 to 80000:1.
 46. The delivery vehicleaccording to claim 6, wherein the combination of the weak acid salt andthe polyprotic acid is at a molar ratio ranging from 8:1 to 200:1. 47.The delivery vehicle according to claim 1, wherein the weak acid salt issodium acetate, potassium acetate or calcium acetate.
 48. The deliveryvehicle according to claim 6, wherein the weak acid salt is at aconcentration of at least 100 mM; and typically, between 100 mM and 800mM; preferably between 200 mM and 800 mM; and most preferably between400 mM and 800 mM.
 49. The delivery vehicle according to claim 6,wherein the liposome comprises a lipid mixture; and the lipid mixturecontains one or more lipids and a polyethylene glycol-derived lipid. 50.The liposomal composition according to claim 34, wherein the mild acidicagent is arachidonic acid metabolite.
 51. The liposomal compositionaccording to claim 34, wherein the mild acidic agent is a prostaglandin.52. A liposomal composition, comprising a delivery vehicle, saiddelivery vehicle comprising a liposome in an aqueous medium, wherein theliposome comprises a combination of a weak acid salt and an organictribasic acid encapsulated within the liposome, wherein the weak acidsalt is sodium acetate, potassium acetate or calcium acetate; and anarachidonic acid metabolite.
 53. The liposomal composition according toclaim 52, wherein the molar ratio of the arachidonic acid metabolite tothe amount of lipids of the liposomal composition is at least 0.001:1.